Porcine circovirus (PCV) was identified in 1974 as a picornavirus-like contaminant of a pig kidney tissue culture cell line (PK15) (1). In 1998, an antigenically and genetically distinct PCV was isolated from pig tissue and named Porcine circovirus type 2 (PCV2); PCV2 is associated with clinical disease in pigs (2).
Postweaning multisystemic wasting syndrome (PMWS), an emerging and economically important disease in swine, is caused by PCV2. However, evidence suggests that manifestation of the signs of PMWS requires co-infection with a pathogen such as Porcine parvovirus (PPV) or a similar immune stimulant, stress, or cofactor. This syndrome debilitates swine 7 to 15 weeks of age, with wasting, respiratory distress, enlarged lymph nodes, diarrhea, pallor, and jaundice. Also, gross and histologic lesions can affect multiple organ systems and are associated with interstitial pneumonia, lymphadenopathy, hepatitis, nephritis, myocarditis, enteritis, dermatitis, and pancreatitis (1,3).
Antibodies specific for PCV2 have been retrospectively detected in swine serum dating back to 1973 (4). Diagnosis of PMWS relies on the detection of either PCV2-specific nucleic acid or antigen associated with lesions in affected tissues. The virus has been isolated from heart, lung, liver, kidney, spleen, salivary gland, lymph node, thyroid, thymus, gastrointestinal tract, feces, pancreas, testes, and brain (1,5).
The primary route of transmission is unknown, but evidence suggests that PCV2 can be transmitted both horizontally and vertically. It has been detected in ocular, nasal, and fecal samples from naturally infected swine. Isolation of PCV2 from aborted pig fetal tissue suggests vertical transmission. Detection of PCV2 nucleic acid in the semen of naturally and experimentally infected boars suggests transmission from boars to PCV2-naïve gilts and their litters (5).
Porcine circovirus type 2 (PCV2) is a small nonenveloped virus with a circular single-stranded DNA (ssDNA) genome. The genome comprises six open reading frames (ORFs) encoding putative proteins (2) with four ORFs sharing significant homology with analogous ORFs from the nonpathogenic porcine circovirus type 1 (PCV1) (6). Only proteins encoded by ORF1, ORF2, and ORF3 have been detected in PCV2-infected cells. ORF1 encodes the 312 amino acid replicase protein and also gives rise to a 168 amino acid spliced variant, both of which are essential for PCV2 replication (7). The structural capsid protein is encoded by ORF2 (8); and ORF3 encodes a highly conserved protein that is not essential for replication but plays an important role in PCV2-induced apoptosis and pathogenicity (9).
Only the surface-exposed PCV2 ORF2-encoded capsid protein is capable of eliciting a protective antibody response by the B cells of the host's immune system (10). Consistent with that observation, PEPSCAN mapping studies localized clusters of immunodominant B cell epitopes with specificity to PCV2 primarily to areas on the PCV2 capsid protein, at residues 65-87, 113-139, 169-183, and 193-207 (11). In addition to B cell epitopes, immunodominant T cell epitopes, including T helper cell epitopes, are another important factor affecting the antigenicity of a virus. T helper cell epitopes elicit protective T helper cell (Th) responses that signal B cells to produce antibody. In contrast to the localization of B cell epitopes to the capsid protein (11), T cell epitope mapping of PCV2 with 20 mer peptides and lymphocyte proliferation assays indicated that immunodominant Th responses are most consistently localized to epitopes on the nonstructural proteins of ORF1 and ORF3, whereas no linear Th epitopes encoded by ORF2 displayed immunodominance (12).
Presentation of Th determinants to the immune system by a synthetic peptide is a critical factor controlling the immunogenicity of a synthetic peptide. Th epitopes that are immunodominant and promiscuous are highly and broadly reactive in populations of divergent MHC types (13). Thus, the scarcity of immunodominant Th sites on the capsid protein helps to account for the limited immunogenicity of subunit capsid vaccines for PCV2. The immunogenicity of a peptide subunit immunogen can be strengthened by covalent linkage of a targeted B cell epitope to selected foreign promiscuous Th sites, including a promiscuous Th epitope whose genetic responsiveness is enlarged through combinatorial chemistry (14, 15, 16). In addition, the immunopotency of a vaccine in swine and protection of swine from viral infection afforded by the vaccine can be influenced by multiple Th epitopes of the virus, and these can be cross-protective in combination with B cell epitopes from different proteins (17).
There is a need for a low cost highly specific vaccine that protects piglets against PCV2-related diseases such as PMWS. Conventional vaccines for pigs are based on inactivated whole PCV2 virus. However, PCV2 does not replicate to high titers in cell culture, making conventional vaccines costly and of low potency. Alternative vaccines are based on recombinant PCV2 ORF2 antigens. PCV2 ORF2 capsid subunits have been expressed in various expression systems including the baculovirus expression system in insect cells (3,10). In most applications, insect cells which produce the recombinant PCV2 capsid proteins are lysed and formulated into vaccines that are used to vaccinate piglets. Such recombinant antigens frequently contain host cell and vector-encoded antigens thus rendering the vaccine nonspecific and can cause severe untoward immune responses including anaphylaxis.
Another problem with inactivated virus PCV2 vaccines, and with subunit capsid vaccines either as recombinant virus lysates or as purified protein, is their relatively low potencies when used in normal field piglets. They are commonly tested in specific pathogen-free (SPF) piglets or cesarean-derived colostrum deprived (CDCD) piglets because those in the field often have maternally-derived antibodies (MDA). These maternal antibodies, while typically of titers too low and of too short duration to provide protection, are still high enough to interfere with vaccination. This interference by MDA can be partially overcome by administration of a high dose of recombinant ORF2 capsid antigen (18). A recombinant full-length ORF2 antigen was secreted into the medium by the baculovirus expression system disclosed in US2009/0022749A1 (19). This system partially alleviates problems of low yield and purity; however, this system and the other biological expression systems suffer from the inherently limited immunogenicity of full-length PVC2 antigen preparations, and they endure costly problems stemming from non-reproducibility, low yields, and complicated quality control protocols. Thus, it is desirable to develop a PCV2 vaccine through a process of rational design, through which immunogenic peptides are designed and synthesized. Such peptide-based vaccines can be designed to contain capsid-specific B cell epitopes, potent foreign Th epitopes and PCV2 Th epitopes derived from other PCV2 proteins, and used in combination as the key ingredient of a vaccine formulation, to elicit anti-capsid antibody responses that precisely confront PCV2 infection in piglets.
The rational design of immunogenic peptides for a virus vaccine begins with the identification of immunodominant epitopes by epitope mapping. Epitope mapping employs a series of overlapping peptides corresponding to regions of interest on a targeted virus protein to identify sites which participate as immunogenic determinants in interactions with the immune system. Most commonly, epitope mapping employs peptides of relatively short length to precisely detect linear determinants. A fast method of epitope mapping known as “PEPSCAN” is based on the simultaneous synthesis of hundreds of overlapping peptides, coupled to solid supports. The coupled peptides are tested for their abilities to bind antibodies or to stimulate T cell proliferation. This approach is effective in localizing linear B and T cell determinants; however, the immunodominant B cell epitopes of a targeted virus or protein that are essential for vaccine development are usually long high affinity discontinuous epitopes that are difficult to define by the PEPSCAN method (20).
There is a need for the identification of immunogenic PCV2 peptides bearing long discontinuous epitopes which can be chemically synthesized in milligram to kilogram quantities by controlled and reproducible solid-phase peptide synthesis. This controlled commercial scale process for the synthesis of PCV2 capsid immunogens, together with the straightforward means to characterize such peptide products, would provide a framework for the low cost commercial scale manufacture and quality control of PCV vaccine formulations (21).